Extended Data Fig. 1: Labelling efficiency of M2 receptor biosensors.
From: Ligand-specific activation trajectories dictate GPCR signalling in cells
(a) Shown are representative confocal images obtained from the M2R labelling screen. M2R constructs (SP-M2RXXXTAG) incorporating the non-canonical amino acid TCO*K at the indicated positions (amino acid numbering of the native human M2 receptor) could be labelled with Tet-Cy3 on at least 3 independent days of experiments. Labelled M2R constructs that show agonist-induced fluorescence intensity changes (see Fig. 1c), are highlighted in orange. (b) For quantification of labelling efficiencies, M2R constructs, each comprising a C-terminal EGFP, were transiently expressed in HEK-AD cells (SP-M2RXXXTAG-EGFP). The average numbers (N) of counted Tet-Cy3 labelled molecules were obtained using molecular brightness analysis41. The number of Tet-Cy3 labelled molecules was then compared to the number of molecules counted in the EGFP channel. Created in BioRender. Thomas, R. (2025) https://BioRender.com/loxqlqf. (c-i) The average number N of counted Tet-Cy3 labelled molecules (y-axis) and EGFP (x-axis), obtained from approx. 100 images, is plotted in a log-log-scale. Each data point indicates a single HEK-AD cell analysed from overall 3 independent experiments. The data were fitted by linear regression with constraints at X,Y = 0. The fits are shown as slope ± sd. n, number of cells. R2, goodness of fit.
